System and method for washing blood and the like



July l 1969 v c. A. scHLUTz 3,452,924

SYSTEM AND METHOD FOR WASHING BLOOD AND THE LIKE Filed Feb. s, 1965vsheet of 2 INVENTOR. C24/wis Alfa/U72 ATTORNE V SYSTEM AND METHOD FORWASHII-IG BLOOD AND THE LIKE Fild Feb. s, 19e-5 C. A. SCHLUTZ Julyvl,1969 Sheet INVENTOR. (f4/mw Es A62-#1. z/rz United States Patent O U.S.Cl. 233-14 6 Claims ABSTRACT OF THE DISCLOSURE Method and apparatus forcontinuously washing blood or other biological cells in suspension byintroducing said suspension at a plurality of radially spaced apartregions within separate tubes that are readily arrayed and movecentrifugally around a common axis.

This invention relates to a method and apparatus for continuouslywashing blood or other biological cells in suspension by passing asaline or other Washing solution through said suspension undercentrifugation to remove contaminating proteins or other materialsattached to the cells.

The apparatus herein may also be used for washing other types of bothorganic and inorganic particles in various suspending media, as may berequired or necessary.

Heretofore, the washing of blood has been accomplished by a batchprocess in which a vial of sterile blood has added to it a first volumeof saline washing solution, after which `the resultant mixture isvigorously shaken manually to effect mixing, and then inserted into acentrifuge. After the vial has been centrifuged, the saline solutiontogether with some plasma contaminants are withdrawn, and a second freshquantity of saline solution is added. The foregoing process is repeatedat least four times to achieve the desired purification. This entireprocessing takes as much as 'four hours or more. One undesirablebyproduct of this batch process is that the long time of processingcauses the blood cells to exude a number of mucoid substances whichcontaminate the sera to which these cells might be added for absorptivepurposes.

The present invention overcomes these drawbacks of the batch process byteaching a system of balanced hydrodynamic centrifugal fluid columns inwhich the saline washing fluid component is continuously interjectedinto a plurality of centrifugal acceleration regions, so that acounterow of blood cells and washing solution simulate the previousshaking operation, thereby combining the mixing, washing, andcentrifugal separation processes, and reducing the processing time tothe order of one-half hour so as to obviate the exudation of mucoids,and to minimize t-he technician-hours spent in processing the blood.

It has been determined by an exhaustive research program using acentrifugal column of glass beads and packed human erythrocytes with thesupernate solution dyed by a neutral Acriflavin dye, that the optimumwashing of a 50 ml. volume centrifuge tube takes place when the Washingsolution is introduced simultaneously on at least several spaced apartregions of centrifugal acceleration. Subsequent tests with the disclosedgradient chamber apparatus have Verified the effectiveness of theinvention in rapidly removing the unwanted residual plasma proteins.

An additional feature of the invention provides sterile means totransfer the cleansed blood cells from the centrifugal gradient chambersinto a sterile storage container.

3,452,924 Patented July 1, 1969 lCe It is an object of this invention toprovide a method of blood washing which 'will continuously and rapidlyunder sterile conditions pass a large volume of Washing solution incounterflow relation to the blood or other biological cells, and whichwill thereafter permit the sterile transfer of the cleansed cells to astorage container.

Another object of the invention is to provide a design of centrifugalgradient chamber which will permit the Iintroduction at a plurality ofacceleration regions of a mixture of blood and washing solution whilecontinuously withdrawing the effluent.

Still another object of the invention is to provide a hydraulic circuitin which a sterile blood bag, a sterile washing solution reservoir, `apositive displacement pump, a balanced column rotating joint continuousflow centrifuge, a centrifugal gradient washing chamber, and an exhaustduct can be interconnected to effect the efficient Washing of the bloodcells or other biological cells. In such an arrangement, disposablesterile blood bags may be utilized and the complete system may bemaintained in a sterile condition from -the input end to the output end.

A tfurther object is to provide a design of auxiliary sterilizableaccessory ducts for the inlet and outlet tubes of the Iforegoingacceleration gradient washing chamber which rwill expedite the steriletransfer of the processed blood cells to a storage container.

Still other objects and advantages of the invention will be apparentfrom the specification.

The features of novelty which are believed to be characteristic of theinvention are set forth herein and will be best understood, both as totheir fundamental principles and to their particular embodiments, byreference to the specification and accompanying drawings, in which:

rFIGURE 1 is a diagrammatic view of an embodiment of the subject bloodor cell washing system, showing the hydraulic circuit;

FIGURE 2 is a side view, partly in section, ont the centrifugalacceleration gradient chamber of the subject invention;

FIGURE 3 is an enlarged, fragmentary view of the lower portion of theinlet gradient injection or distributor tube, a portion of which isshown in FIGURE 2, and which illustrates the spaced apart distributorports as Well as the restrictive end orifices therein;

FIGURE 4 is a side view showing the installation of the auxiliary intakeair filter and exhaust needle with its guard, preparatory to thetransfer of washed blood to its storage container; and

FIGURE 5 is a somewhat smaller wiew of the foregoing transfer assemblyfor gravity transfer of Washed blood cells to its storage bottle.

Referring to the diagram of FIGURE 1, plastic bag 21 contains thesterile blood specimen, which typically might have a volume of from to400 ml., said bag 21 being suspended at the highest point in the systemto generate a hydrostatic head which insures that the blood be depositedinto the centrifuge early in the washing cycle. Bag 21 is sterilized andevacuated prior to being lled with the blood sample.

A reservoir bottle 22 for the washing solution is provided With astopper 23 through which are sealed a short air intake tube 24 and along deep outlet tube 26. Intake tube 24 communicates with the outsideatmosphere through an air filter 27 to maintain the sterility of thesystem.

The washing solution, which will typically be a hypertonic salinesolution having a volume of four to ve times that of the blood sample,is drawn from the bottom of bottle 22 via tubes 26, 28 and 29, at acontrolled rate by the action of the positive displacement electricmotor driven pump 31. A useful type of pump for this purpose is onewhich uses a pair of spaced apart rotating rollers on a semi-circulartrack where they progressively collapse the wall of resilient rubbertube 29 so as positively to displace the fluid without the possibilityof contaminating it.

Similarly, the blood from bag 21 is drawn by pump 31 via tube 32 and thetubular junction fitting 33, where the blood and saline washing solutionmix, to pass together through pump 31 and tube 29. The mixture thenpasses through a flow meter 34, the series connecting tubes 36, 37 and38, thence entering the electrically motor driven rotating-jointcontinuous iiow centrifuge rotor 39. Such centrifuges, which are wellknown in the art, are based on the principle of balanced hydrodynamiccolumns, in the sense that incoming liquid, such as that entering viatube 38, is passed through a rotating seal into a centrifugal chamber ortube 44 having a closed outer end or bottom through a path which carriesit into progressively higher centrifugal accelerations.

However, on continuous fiow centrifuges, the outlet column also passesthrough a separate rotary seal and hydraulically joins the inlet columnin the centrifuging chamber. Consequently, except for the difference indensity between the incoming and outgoing columns, there is a majorbalancing of the hydrodynamic columns, thereby permitting the continuousflow into and out of the high centrifugal field forces.

The motivating force which causes fiow of fluid into and out of thecentrifugal field is hydraulic head; i.e., a preponderance of fiuid headin the inlet lines over that in the outlet. Thus the incoming fluid isconducted down the rotating axis of the centrifuge to a small centralchamber where it is distributed to all the tubes from a point as nearthe axis of rotation as possible. The outlet tube, on the other hand,feeds to a chamber also concentric with the axis, but whose periphery isradially further away. The difference, in these two resultant radialcolumns, then, is the hydraulic head which effects continuous iiowthrough the rotating centrifuge rotor.

To maintain balance, the centrifuge tubes 44 are used in diametricallyopposite pairs, and by a double manifold (not shown) the inlet tubes 48of the several tubes 44 are connected together, and the outlet tubes 49of these tubes are connected together.

The effluent from the continuous flow centrifuge 39 is passed viaserially connected tubes 41, 42 and 43, to a waste discharge. The entirecentrifuge 39 together with its motor 46 is housed within a temperaturecontrolled chamber 47 which may also be maintained in sterile condition.

Centrifuge tubes 44 extend radially outwardly and downwardly from theaxis of rotation of rotor 39. As said tubes 44 move around said axis ofrotation, centrifugal force fields having radial gradients ofacceleration are set up in said tubes, the centrifugal forces increasingin different region of said tubes depending upon their distances thereinfrom said axis of rotation.

Referring now to FIGURE 2, which shows the detailed construction of thesubject acceleration gradient centrifuge tube 44, there is provided acircular compression element 51 having a central upwardly extendingthreaded shaft 52 which extends freely through a central aperture incircular cap 53 which extends over and abuts the upper end of centrifugetube 44. Cap 53 has an annular collar 54 which extends inwardly into thechamber of tube 44. Compression element 51 has an annular recess whichaccommodates a resilient O-ring 55. When nut 56 is tightened on shaft52, O-ring 55 is expanded by the increased pressure between compressionelement 51 and collar 54 to effect a seal between the inner wall ofcentrifuge tube 44 and the assembly of said compression element and cap.

Both inlet tube 48 and outlet tube 49 are secured and sealed to thecompression element 51 and extend freely through suitable apertures, notshown, in cap 53. Outlet tube 49 terminates flush with the inner surfaceof element 51 while inlet tube 48 continues nearly to the bottom end ofthe chamber of centrifuge tube 44.

Inlet tube 48 is constricted at its bottom end to form one or two smallterminal ports 57 (see FIGURE 3). In addition, tube 48 is provided withgroups of spaced apart radial distributor ports 58.

Distributor ports 58 are not only located in spaced apart gradients ofcentrifugal fields, but their respective locations also facilitate gooddispersion and mixing of the wash fluid with the specimen cells.Depending upon the type and size of cells and other particulatematerials that are to be washed and upon the nature of the washingmedium, the sizes of distributor ports 58 may be selected and determinedto provide for optimum efiiciency of the washing process. The size ofdistributor ports 58 may also be selected and determined to control therate of dispersion and velocity at which the materials flow through tube44.

In one embodiment, the dimensions of the holes 58 are approximatelydrill size 60, but it is contemplated that other drill sizes may beselected for said holes in adapting the apparatus for different types ofmaterials to be processed thereby.

By making ports 57 small enough so that fiow therethrough iscomparatively slow, a back pressure is induced throughout inlet tube 48thereby causing the incoming mixture of blood and washing fluid to flowthrough all of the ports 58 along the length of said tube into severalspaced-apart regions of centrifuge tube 44. By this means, during thecentrifuging motion of centrifuge tubes 48, the heavier blood cells willmove outwardly and downwardly, while the washing fiuid which is oflesser density than the blood cells will move in a counterfiow directioninwardly and upwardly through centrifuge tube 44 and emerge as aneffluent through outlet tube 49 to be transmitted through tubes 41, 42,43 to a discard location.

The provision of a plurality of regions of introduction of the materialsto 'be centrifuged greatly increases the efficiency of the washingaction taking place within the centrifuge tube 44 and greatly speeds upthe production of washed cells on a continuous basis.

After the washing operation is complete, and before the washing uid inbottle 22 reaches the bottom of tube 26, the centrifuge motor 46 isdeenergized to stop the rotor and the centrifuge tubes 44 are removed.

In order to transfer the processed blood cells from tube 44 to a storagebottle 59 (FIGURE 5), an air filter 61 (FIGURE 4) is attached by asterile tube 62 to tube 48 while a hollow sterile needle 63 with aprotective cover 64 is connected to tube 49 by the sterile tube 66.

Tube 44 is then clamped in a support pedestal 67, cover 64 is removed,and the needle 63 is caused to pierce the resilient cap 68 enclosingstorage bottle 59. Gravity will then complete the transfer.

In actual experience with the apparatus described herein, sterileconditions have been achieved from the input end to the output end ofthe system as exemplified by passing twenty liters of thioglycollatemedia through the system on several occasions with sterile salvage. Theefficiency of the wash system has been established as determined byfindings of removal of human protein when tested by anti-humanprecipitant serums.

It is to be noted that the principles of this invention may also be usedfor the washing of bacterial germ cells in various liquid agents, and inthe glycerolization of red cells.

I claim:

1. The method of washing blood cells or the like in a liquid washingsolution comprising the steps of forming a mixture of blood with aliquid washing solution having less density than said blood, moving aradially extending centrifuge tube with a closed outer end around anaxis of rotation, introducing said mixture into said moving centrifugetube at a plurality of radially spaced apart regions therethrough havingdiffering values of centrifugal acceleration whereby the blood cells insaid blood move in an outward direction in said centrifuge tube incounterflow to the movement of said washing solution, and withdrawingthe effluent washing liquid from said centrifuge tube at a region ofleast centrifugal acceleration.

2. A method according to claim 1 and further cornprising means forcontinuously transmitting said mixture into said centrifuge tube as itrotates, and means for continuously removing the efuent washing liquidfrom said centrifuge tube.

3. A centrifuge structure for washing biological cells such as blood orthe like comprising a rotor, a plurality of centrifuge tubes havingclosed outer ends arrayed radially in said rotor, means for continuouslyintroducing materials to be centrifuged into each of said centrifugetubes as the latter move around the axis of rotat-ion of said rotor, andoutlet tube for continuously removing eluents from said centrifuge tubesduring said rotation, said introducing means comprising an inlet tubeextending into the interior and substantially throughout the length ofeach centrifuge tube and a plurality of spaced apart ports on said inlettube for introducing said materials into the respective centrifuge tubeat spaced apart regions therethrough where different degrees ofcentrifugal acceleration are engendered during rotation of said rotor.

4. A structure according to claim 3 and further comprising aconstriction on the outer end of said inlet tube in the interior of saidcentrifuge tube, said constriction being suficiently small to induceback pressure in said inlet tube to cause the materials to becentrifuged to emerge simultaneously through all of the spaced apartports therein.

5. A structure according to claim 3 and further comprising aconstriction on the end of said inlet tube in the interior of saidcentrifuge tube, said constriction forming at least one port on the endof said inlet tube, the aperture of said port being sufficiently smallto induce back pressure in said inlet tube to cause said materials toemerge simultaneously through all of the spaced apart ports on theremainder of said inlet tube.

6. A continuous Washing system for biological cells in suspensionaccording to claim 3 and further comprising a container for saidsuspension, a washing solution container', an air inlet duct includingan air lter communicating with said washing solution container, apositive displacement pump, first duct means communicating between theinlet of said pump and both said suspension container and said solutioncontainer, second duct means communicating between the output of saidpump and the said inlet tube of said centrifuge tube, and third ductmeans communicating between said outlet tube of said centrifuge tube andthe exterior of said centrifuge.

References Cited UNITED STATES PATENTS 2,834,541 5/ 1958 Szent-Gyorgy etal. 233-26 3,050,238 8/1962 Doyle et al 233-15 3,133,880 5/1964 Madany233-15 3,235,173 2/1966 Unger 233-26 XR 2,758,597 8/1956 Elder 12S-2142,775,240 12/ 1956 Morrissey et al. 128-214 3,064,646 11/ 1962 Earl128-272 XR4 3,185,154 5/1965 Caccavo et al. 128-272 3,347,454 10/1967Bellamy et al. 233-26 XR 3,092,106 6/ 1963 Butler 128-214 FOREIGNPATENTS 864,410 4/ 1961 `Great Britain.

HENRY T. KLINKSIEK, Primary Examiner.

U.S. Cl. X.R.

